Allosteric modulation balances thermodynamic stability and restores function of ΔF508 CFTR

Abstract

Most cystic fibrosis is caused by a deletion of a single residue (F508) in CFTR (cystic fibrosis transmembrane conductance regulator) that disrupts the folding and biosynthetic maturation of the ion channel protein. Progress towards understanding the underlying mechanisms and overcoming the defect remains incomplete. Here, we show that the thermal instability of human ΔF508 CFTR channel activity evident in both cell-attached membrane patches and planar phospholipid bilayers is not observed in corresponding mutant CFTRs of several non-mammalian species. These more stable orthologs are distinguished from their mammalian counterparts by the substitution of proline residues at several key dynamic locations in first N-terminal nucleotide-binding domain (NBD1), including the structurally diverse region, the γ-phosphate switch loop, and the regulatory insertion. Molecular dynamics analyses revealed that addition of the prolines could reduce flexibility at these locations and increase the temperatures of unfolding transitions of ΔF508 NBD1 to that of the wild type. Introduction of these prolines experimentally into full-length human ΔF508 CFTR together with the already recognized I539T suppressor mutation, also in the structurally diverse region, restored channel function and thermodynamic stability as well as its trafficking to and lifetime at the cell surface. Thus, while cellular manipulations that circumvent its culling by quality control systems leave ΔF508 CFTR dysfunctional at physiological temperature, restoration of the delicate balance between the dynamic protein's inherent stability and channel activity returns a near-normal state.

Figure 1

Corrector plus low temperature rescued human ΔF508 CFTR in cell-attached patches is thermo-labile. (a) Western blot showing formation of a slower migrating diffuse mature band in BHK cells stably expressing ΔF508 CFTR mutant grown at 27°C ((r)ΔF) without (left lane) or with (right lane) exposure overnight to correctors VRT-325 and Corr-4a at 10uM. Positions of immature and mature bands are shown on the left by open and filled arrowheads respectively. (b) Cell-attached recordings of the wild type CFTR (WT) ion channels beginning at 27°C and then where indicated, ramped up to 32°C at a rate of 1°C/10 sec. Prior to recording, cells were perfused with a “cation-free” solution containing 150 mM Tris/HCl, pH 7.2; 1 mM EGTA and 1 g/l glucose supplemented with 15 µM forskolin to activate CFTR ion channel function. The upper level in each trace represents overall closed state for all channels while downward deflections correspond to channel openings and each next lower level represents one more channel opening. (c) Cell-attached recordings of ΔF508 CFTR rescued by low temperature and correctors ((r)ΔF) with the same experimental protocol as in (b). X axis scale bar – 4s and Y axis scale bar – 1 pA for both (b) and (c) panels. (d) Since the gating kinetics of the ΔF508 CFTR is unstable the bar graph of mean conductance of single structural units <γ> that we consider as a measure of functional capacity for the WT and (r)ΔF at +27°C and 32°C is shown instead of probability to be open (P_o). For WT CFTR <γ> was obtained from continuous 100 s recordings at each temperature and normalized to the number of channels in the patch while for (r)ΔF it was calculated from 300 s of recordings at 27°C and the following 180 s at 32°C and normalized. In cell-attached configuration the single channel chord conductance of both wild type and temperature rescued ΔF508 CFTR was estimated as 8.5 pS at 27°C and 11.3 pS at 32°C. Asterisk indicates significant differences from wild type CFTR (p < 0.05).

Figure 2

Maturation and life-times of WT and ΔF508 CFTRs from different species. (a) Western blots showing relative amounts of smaller immature and larger mature bands. Cell growth temperatures and genotypes are indicated above lanes. C-terminal GFP tags are present in all species except human accounting for their slower mobilities. Faster mobility of shark bands is due to glycosylation of 1 rather than 2 sites. 7.5% acrylamide SDS-PAGE blot probed with mAb 596. (b) Chicken CFTR short-term pulse-chase experiments with ³⁵S-methionine (20 minute pulse; chase in hours). Autoradiograms shown were also quantified electronically (Packard Instant Imager) and rates of disappearance of immature precursors and appearance of mature products graphed. Symbols: human wild-type in red; chicken wild-type in blue; chicken ΔF509 in green. (c) Long-term pulse-chase experiments showing rates of turnover of mature species. Experimental data shown on the graph are mean value +SEM from 3 independent experiments.

Figure 3

WT and ΔF509 chicken CFTR have similar ATP binding and ion channel activities. (a) Membranes containing human wild-type (WT), temperature rescued human ΔF508 ((r)ΔF), chicken wild-type (ChWT) or chicken ΔF509 (ChΔF) CFTR were incubated with 25 µM [γ³²P]-8N₃ATP at 4°C or 35°C (left panels) and irradiated at 254 nm for 2 minutes and digested with trypsin (10 µg/ml) for 15 minutes at 4°C. Following this limited proteolysis, membranes were solubilized in RIPA buffer, immunoprecipitated with an antibody recognizing an N-terminal epitope (mAb13-4), subjected to SDS-PAGE (4–20% acrylamide gradient gels) and transferred to nitrocellulose for autoradiographic detection of ³²P radioactivity. In the right panels, the labeled membranes were (+) or were not (−) washed free of bulk nucleotide before solubilization. (b) Iodide efflux from BHK-21 cells stably expressing WT human CFTR in red, WT chicken CFTR in blue, ΔF509 chicken CFTR in green and human ΔF508CFTR in purple. After iodide loading, extracellular iodide was removed by rinsing the cells with iodide-free efflux buffer (same as the loading buffer except NaNO₃ replaced NaI). Samples were collected by replacing the efflux buffer (1 ml volume) with fresh solution at 1 min intervals. The first four samples were used to establish the baseline. Stimulation cocktail with PKA agonists (10 µM forskolin, 100 µM dibutyl-cAMP and 1 mM 3-isobuty-1-methylxanthine) was added and iodide efflux was measured using an iodide selective electrode. At the end of each assay, efflux buffer containing 0.1% NP-40 was added (shown by arrow) to release any retained iodide. Experimental data are shown as mean values ± SEM from at least 3 independent experiments.(c) Single channel activities also at 25°C.The upper level in each trace represents closed state and downward deflections corresponds to channel opening. All-points histograms fitted by two-peak Gaussians (red line) are to the left of each tracing and single channel conductance (γ) and open probabilities (P_o) above. X axis scale bar – 10s.

Figure 4

Avian ΔF509 CFTR channel is nearly as active and thermally stable as the WT. (a) Wild-type chicken CFTR(ChWT) single channel tracings and all-point histograms fitted by two-peak Gaussians (red line) at the temperatures indicated. X axis scale bar – 10s. (b) ΔF509 chickenCFTR (ChΔF) single channel current recorded at the same temperatures. X axis scale bar – 10s. (c) Chicken (blue line and symbols) and human (red line and symbols) CFTR single channel conductance as a function of temperature. Data shown as mean values ± SEM. (d) Plots of the transport capacity of the structural unit <γ> for chicken wild-type (blue), chickenΔF509 CFTR (green) at different temperatures compared to human wild-type (red) and human ΔF508 CFTR (purple). Data are shown as mean value with symbol sizes that exceed SEM.

Figure 5

Mimicking chicken CFTR (prolines and I539T) restores maturation and lifetime of ΔF508 CFTR. (a) Pattern of proline substitutions in NBD1 of CFTR orthologs with different sensitivities to the ΔF508 mutation. Tabulated are residue positions (human amino acid numbering) where prolines commonly replace other residues in the non-mammalian species in which ΔF508 variants mature at 37°C and are stable. The common I539T replacement also is indicated. (b) NBD1 3D structure (PDB 2BBO) is shown with positions of tested residues indicated. RI is colored red, SDR is colored orange and the F508-loop is colored green. (c) Western blot of wild-type and ΔF508 CFTR expressed in HEK-293 cells and ΔF508 modified with I539T (ΔF/T), S422P/S434P/S492P/A534P (ΔF/4P), I539T/S492P (ΔF/PT), I539T/S492P/A534P (ΔF/2PT) and I539T/S422P/S434P/S492P/A534P (ΔF/4PT). Methods as in Figure 2(A). (d) Short-term pulse-chase experiments with variants indicated. Qualitative and quantitative autoradiograms as in Figure 2b. Color code: ΔF/T – red, ΔF/PT – orange, ΔF/2PT – green, ΔF/4PT – blue, human WT – black. (e) Long-term pulse-chase experiments performed and analyzed as in Figure 2c and shown with the same colors as in (b).

Figure 6

Proline substitutions reveal trade-off between thermostability and channel activity of ΔF508 CFTR. (a) Single channel tracings of ΔF508 CFTR with the substitutions indicated. All recordings prepared at 35°C. All point histograms fitted by two-peak Gaussians (red line) are shown on the left of each tracing. X axis scale bar 10s. (b) Gating of ΔF508 CFTRs with substitutions indicated during a continuous temperature ramp of 1°C/minute. Temperature was maintained at 35°C for 3 minutes before initiating the ramp and held at 40°C for 2 minutes after this temperature was reached. X axis scale bar 60s, Y axis scale bar 1 pA. (c) Plots of the transport capacity of the structural unit. These <γ> values were calculated after 10 min incubation at each of the five temperatures indicated to obtain equilibrium values. See Methods for the details of <γ> calculations for the transport unit with unstable open state.

Figure 7

Restoration of ΔF508 CFTR NBD1-CL4 interface by proline and I539T mutations. HEK293 cells were transiently transfected with Cys-less CFTR or Cys-less ΔF508-CFTR in the presence or absence of the 4PT mutations (S422P/S434P/S492P/A534P/I539T), with the Cys pair V510C/G1069C introduced at the CL4/NBD1 interface. 48 hrs after transfection, cells were incubated with 200 µM M1M or M8M and cell lysates in SDS-PAGE sample buffer with or without DTT as indicated were subjected to Western blot analysis with mAb 596. Red arrowhead indicates cross-linked CFTR; solid arrowhead indicates mature complex-glycosylated CFTR and open arrowhead indicates immature core-glycosylated CFTR.

Figure 8

Avian ΔF509 CFTR is destabilized by reversal of proline and I539T substitutions. (a) Western blot of chicken ΔF509 CFTR showing the influence of the “humanizing” T540I and P493S/P535A substitutions. Cells expressing these variants were grown at the temperatures indicated. (b) Single channel activity at +35°C of chicken T540I CFTR substitution (upper tracing), the temperature rescued chicken ΔF509 CFTR with either the T540I (middle tracing) or the P493S/P535A (lower tracing) substitutions. X axis scale bar - 10s, Y-axis scale bar – 1 pA.

Figure 9

Stabilization of the SDR with increasing proline substitutions. (a) Tube representation of different NBD1 constructs. The thickness of the tube is proportional to the average root mean square fluctuation (RMSF) of the corresponding residue during simulations. Rainbow colors are assigned to residues based on their RMSF values with blue representing low RMSF and red representing high RMSF. Different regions of NBD1 that influence the dynamics of the domain are marked for WT NBD1. The remaining panels are oriented in the same way as WT NBD1 for simplicity. (b) Cv profile for different NBD1 constructs showing increase in the folding transition temperature with increase in the number of proline substitutions. Peaks in the plot represent structural transitions in NBD1. Dashed lines indicate the temperatures, black: ~328 K, red: ~313 K, orange: ~327 K, green: ~326 K, blue: ~335 K, at which major structural transitions take place in the corresponding constructs. See results and discussion for further details.